AU2005263993B2

AU2005263993B2 - Cell cycle reporting cell line

Info

Publication number: AU2005263993B2
Application number: AU2005263993A
Authority: AU
Inventors: Ellen Fanning; Jinming Gu; Suzanne Hancock; Simon Stubbs; Nicholas Thomas
Original assignee: GE Healthcare UK Ltd; Vanderbilt University
Current assignee: GE Healthcare UK Ltd; Vanderbilt University
Priority date: 2004-07-23
Filing date: 2005-07-22
Publication date: 2009-11-12
Anticipated expiration: 2025-07-22
Also published as: JP5080252B2; US20100028899A1; JP2008511289A; US7897741B2; WO2006008542A1; AU2005263988A1; DE602005021493D1; ATE469172T1; JP5143552B2; EP1771465A1; AU2005263988B2; CA2574530A1; US20070264681A1; EP1771465B1; EP1771466B1; WO2006008547A1; AU2005263993A1; US7612189B2; CA2574540A1; EP1771466A1

Abstract

The present invention relates to polypeptide and nucleic acids constructs which are useful for determining the cell cycle status of a mammalian cell. Host cells transfected with these nucleic acid constructs can be used to determine the effects that test agents have upon the mammalian cell cycle.

Description

WO 2006/008547 PCT/GB2005/002890 CELL CYCLE REPORTING CELL LINE Technical Field The present invention relates to novel non-destructive and dynamic 5 means for determining the cell cycle position of living cells. Background of the Invention Eukaryotic cell division proceeds through a highly regulated cell cycle comprising consecutive phases termed G1, S, G2 and M. The two transition 10 phases, G1 and G2, are interspersed between the DNA synthesis (S) phase in which cellular DNA is replicated and the mitosis (M) phase in which each cell divides to form two daughter cells. Disruption of the cell cycle or cell cycle control can result in cellular 15 abnormalities or disease states such as cancer which arise from multiple genetic changes that transform growth-limited cells into highly invasive cells that are unresponsive to normal control of growth. Transition of normal cells into cancer cells arises though loss of correct function in DNA replication and DNA repair mechanisms. All dividing cells are subject to a number of control 20 mechanisms, known as cell-cycle checkpoints, which maintain genomic integrity by arresting or inducing destruction of aberrant cells. Investigation of cell cycle progression and control is consequently of significant interest in designing anticancer drugs (Flatt PM and Pietenpol JA 2000 Drug Metab Rev 32(3-4):283-305; Buolamwini JK 2000 Current Pharmaceutical Design 6, 379 25 392). Accurate determination of cell cycle status is a key requirement for investigation of cellular processes which affect the cell cycle or are dependent on cell cycle position. Such measurements are particularly vital in drug 30 screening applications where: a) drugs which directly or indirectly modify cell cycle progression are desired, for example as anti-cancer treatments.

WO 2006/008547 PCT/GB2005/002890 b) drugs are to be checked for unwanted effects on cell cycle progression. c) it is suspected that an agent is active or inactive towards cells in a particular phase of the cell cycle. 5 Traditionally cell cycle status for cell populations has been determined by flow cytometry using fluorescent dyes which stain the DNA content of cell nuclei (Barlogie B etal. Cancer Res. 1983 43(9):3982-97). Flow cytometry yields quantitative information on the DNA content of cells and hence allows determination of the relative numbers of cells in, or the proportion of cells 10 in,the G1, S and G2+M phases of the cell cycle. However this analysis is a destructive non-dynamic process and requires serial sampling of a population to determine cell cycle status with time. Furthermore standard flow cytometry techniques examine only the total cell population in the sample and do not yield data on individual cells which precludes study of cell cycle status of 15 different cell types that may be present within the sample under analysis. Flow cytometry is therefore suitable for examining the overall cell cycle distribution of cells within a population but cannot be used to monitor the precise cell cycle status of an individual cell over time. 20 Consequently what is needed to study the effects of agents with desired or undesired effects on the cell cycle is a method to precisely determine cell cycle status of a single living cell by a non-destructive method that allows the same cell to be repeatedly interrogated over time. Furthermore it would be advantageous for cell cycle position to be determined from a probe 25 controlled directly by cell cycle control components, rather than indirectly through DNA content or other indirect markers of cell cycle position as described above. A number of methods have been described which make use of certain 30 components of the cell cycle control mechanisms to provide procedures which analyse or exploit cell proliferation status. 2 WO 2006/008547 PCT/GB2005/002890 US 6048693 describes a method for screening for compounds affecting cell cycle regulatory proteins wherein expression of a reporter gene is linked to control elements which are acted on by cyclins or other cell cycle control proteins. In this method temporal expression of a reporter gene product is 5 driven in a cell cycle specific fashion and compounds acting on one or more cell cycle control components may increase or decrease expression levels. Since the assay system contains no elements which provide for the destruction of the reporter gene product nor for destruction of any signal arising from the reporter gene, the method cannot yield information on the cell 10 cycle position of any cells in the assay and reports only on general perturbations of cell cycle control mechanisms. WO 03/031612 describes DNA reporter constructs and methods for determining the cell cycle position of living mammalian cells by means 15 of cell cycle phase-specific expression control elements and destruction control elements. One embodiment uses well characterised elements of the cell cycle control protein Cyclin B1 to control the expression and degradation of a green fluorescent protein (GFP) molecule to report cellular transition through G2 and M phases of the cell cycle. Since this 20 construct is under the control of the Cyclin B1 promoter, GFP expression is absent during G1 and S thus preventing analysis of cells in these phases of the cell cycle. A human helicase B homolog has been reported and 25 characterised ((Taneja et al J. Biol. Chem., (2002), 277, 40853-40861). The report demonstrates that helicase activity is needed during G1 to promote the G1/S transition. Gu et al (Mol. Biol. Cell., (2004), 15, 3320-3332) have shown that a 30 small C-terminal region of the helicase B gene termed the phosphorylation-dependent subcellular localization domain (PSLD) is 3 WO 2006/008547 PCT/GB2005/002890 phosphorylated by Cdk2/cyclin E and contains NLS and NES sequences. Gu et al (Mol. Biol. Cell., (2004), 15, 3320-3332) carried out studies on cells that had been transiently transfected with plasmid encoding an EGFP-fiGal-PSLD fusion (beta-galactosidase (fiGal) was included in the 5 construct as an inert group to make the whole fusion protein similar in size to the complete helicase B) expressed from a CMV promoter. Cells in G1 exhibited EGFP signal predominantly in the nucleus, whilst cells in other phases of the cell cycle exhibited predominantly cytoplasmic EGFP signal. These researchers concluded that the PSLD was directing 10 translocation of the fiGal-EGFP reporter from the nucleus to the cytoplasm around the G1/S phase transition of the cell cycle. None of the preceding methods which use components of the cell cycle control mechanism provides means for readily and accurately determining the 15 cell cycle status of an individual cell or a population of cells throughout the entire cell cycle. Accordingly a method has been developed and is herein described which uses key components of the cell cycle regulatory machinery, in defined combinations, to drive dual independent cellular reporters to provide novel means of determining cell cycle status at all phases of the cell 20 cycle in individual living cells. Summary of the Invention According to the first aspect of the invention, there is provided a stable cell line expressing: 25 i) a first polypeptide construct comprising a first detectable live-cell reporter molecule linked to at least one cell cycle phase-dependent location control element, the location of which construct within a mammalian cell is indicative of the cell cycle position; and ii) a second polypeptide construct comprising a second detectable live 30 cell reporter molecule linked to a destruction control element wherein said second reporter is detectable in a mammalian cell at a predetermined position in the cell cycle, 4 WO 2006/008547 PCT/GB2005/002890 wherein said first and second reporter molecules are distinguishable from each other and the stable cell line can be used to determine the cell cycle position. 5 The present invention provides cell lines containing polypeptide constructs which exhibit cell cycle phase specific activation, translocation or destruction of detectable reporter molecules by direct linkage of reporter signals to temporal and spatial expression, localisation and destruction of cell cycle components. This greatly improves the precision of determination of cell 10 cycle phase status and allows continuous monitoring of cell cycle progression in individual cells. Furthermore the inventors have discovered that these key control elements can be isolated and abstracted from functional elements of the cell cycle control mechanism to permit design of cell cycle phase reporters which are dynamically regulated and operate in concert with, but 15 independently of, endogenous cell cycle control components and hence provide means for monitoring cell cycle status without influencing or interfering with the natural progression of the cell cycle. Suitably, the cell cycle phase-dependent location control element is 20 selected from the group of peptides consisting of Rag2, Chaf1B, Fen1, PPP1R2, helicase B, sgk, CDC6 or motifs therein such as the phosphorylation-dependent subcellular localization domain of the C terminal special control region of helicase B (PSLD). 25 Suitably, the destruction control element comprises the Cyclin B1 D box. Suitably, the first and second live-cell reporter molecules are selected from the group consisting of fluorescent protein and enzyme reporter. 30 Preferably, said fluorescent protein is selected from the group consisting of Green Fluorescent Protein (GFP), Enhanced Green Fluorescent 5 WO 2006/008547 PCT/GB2005/002890 Protein (EGFP), Emerald and J-Red. Preferably, said enzyme reporter is halo-tag (Promega). Preferably, the first reporter molecule is EGFP and the second reporter 5 molecule is J-Red, or the first reporter molecule is J-Red and the second reporter molecule is EGFP. More preferably, the first reporter molecule is J-Red and the second reporter molecule is EGFP. In one preferred embodiment of the invention the first polypeptide 10 construct comprises the phosphorylation-dependent subcellular localization domain of the C-terminal spatial control region of helicase B (PSLD) coupled to a red fluorescent protein (RFP), and the second polypeptide construct comprises 171 amino acids of the amino terminus of cyclin B1 coupled to a green fluorescent protein (GFP) expressed under the control of the cyclin B1 15 promoter. When expressed in a mammalian cell these constructs exhibit cell cycle specific expression and destruction of the GFP construct and translocation of the RFP construct, with the GFP construct paralleling the 20 expression and degradation of endogenous cyclin B1, and the RFP construct paralleling the translocation of endogenous Helicase B. Hence measurement of both GFP and RFP fluorescence intensity and localisation permits identification of cells in G1, S, G2 and M phases of the cell cycle. Analysis of the fluorescence characteristics of each cell in a population with time 25 consequently yields dynamic information on the cell cycle status of each cell. In further aspects of the invention there are provided methods for analysing cell cycle distribution of cultured cells and determining the effects of test agents on cell cycle distribution. The term 'test agent' should be 30 construed as a form of electromagnetic radiation or as a chemical entity. Preferably, the test agent is a chemical entity selected from the group consisting of drug, nucleic acid, hormone, protein and peptide. The test 6 WO 2006/008547 PCT/GB2005/002890 agent may be applied exogenously to the cell or may be a peptide or protein that is expressed in the cell under study. Thus, in a second aspect of the present invention, there is provided a 5 method for determining the cell cycle position of a mammalian cell, said method comprising: a) culturing a stable cell line as hereinbefore described; and b) determining the cell cycle position by monitoring signals emitted by the first and second reporter molecules. 10 In a third aspect of the present invention, there is provided a method of determining the effect of a test agent on the cell cycle position of a mammalian cell, said method comprising: a) culturing a stable cell line as hereinbefore described; and 15 b) determining the cell cycle position by monitoring signals emitted by the first and second reporter molecules wherein a difference between the emitted signals measured in the absence and in the presence of said test agent is indicative of the effect of the test agent on the cell cycle position of the cell. 20 In a fourth aspect of the present invention, there is provided a method of determining the effect of a test agent on the cell cycle position of a mammalian cell, said method comprising: a) culturing a stable cell line as hereinbefore described; 25 b) determining the cell cycle position by monitoring signals emitted by the first and second reporter molecules; and c) comparing the emitted signals in the presence of the test agent with a known value for the emitted signals in the absence of the test agent; wherein a difference between the emitted signals measured in the presence of 30 the test agent and said known value in the absence of the test agent is indicative of the effect of the test agent on the cell cycle position of the cell. 7 WO 2006/008547 PCT/GB2005/002890 In yet a further aspect of the present invention methods are provided for determining the cell cycle dependencies of cellular processes by means of monitoring cellular processes in cells reporting the cell cycle position. 5 Thus, according to the fifth aspect of the present invention, there is provided a method of determining the effect of the mammalian cell cycle on a cellular process monitored by a process reporter which is known to vary in response to a test agent, said method comprising: a) culturing a stable cell line as hereinbefore described; 10 b) determining the cell cycle position by monitoring signals emitted by the first and second reporter molecules; and c) monitoring the signals emitted by the process reporter wherein the process reporter is distinguishable from the first and second reporter molecules; 15 wherein the relationship between cell cycle position determined by step b) and the signal emitted by the process reporter is indicative of whether or not the cellular process is cell cycle dependent. Brief Description of the Drawings 20 SEQ ID NO: 1 is the amino acid sequence of the PSLD - J-RED fusion protein. SEQ ID NO: 2 is the nucleic acid sequence for pCORON1022-JRed-C1-PSLD. SEQ ID NO: 3 is the nucleic acid sequence for pCORON1020-JRed-C1-PSLD 25 Figure 1 is a vector map of pCORON1002-EGFP-C1-PSLD. Figure 2 shows vector maps of pCORON1022-JRed-C1-PSLD (Figure 2a) and pCORON1020-JRed-C1-PSLD (Figure 2b). 30 Figure 3 presents images of U2OS cells expressing the cyclin BI-EGFP and the PSLD-J Red fluorescent proteins using an IN Cell Analyzer 1000 (GE 8 WO 2006/008547 PCT/GB2005/002890 Healthcare) imaging system. The cells are seen to be at varying stages of their cell cycle, as depicted by letters/numerals 'S', 'G1' and 'G2'. The presence of the Hoechst dye is indicated by the blue fluorescence in Figure 3a, of expression of the G2/M Cyclin B1 green fluorescent protein reporter in 5 Figure 3b and the expression of the G1/S PSLD red fluorescent protein reporter in Figure 3c. Detailed Description of the Invention PSLD-RFP Construct 10 Full-length Human DNA helicase B (HDHB) cDNA was inserted as a BgllI/Notl fragment (Taneja et al., J. Biol. Chem., (2002) 277, 40853-40861) into the Notl site of the pEGFP-C1 vector (Clontech). PCR amplification of the 390 bp PSLD region and introduction of 5' Nhel and 3' SaIl restriction enzyme 15 sites to the PSLD fragment were used to sub-clone into the vector pCORON1002-EGFP-C1 (GE Healthcare). The resulting 6704 bp DNA construct pCORON1002-EGFP-C1-PSLD (Figure 1), contains an ubiquitin C promoter, a bacterial ampicillin resistance gene and a mammalian neomycin resistance gene. Further modification of this vector was carried out using 20 standard PCR and cloning techiques (Sambrook, J. et al (1989)) to replace the EGFP with the fluorescent protein J-Red (Evrogen), to convert the plasmid from the neomycin resistance to hygromycin resistance (Figure 2a) and to replace the ubiquitin C promoter with CMV IE/promoter (Figure 2b). 25 Dual Construct Stable Cell Line A U2OS cell line stably expressing a Cyclin BI-EGFP cell cycle reporter (as described in W003/031612 and supplied under product code 25-80-10 'G2M Cell Cycle Phase Marker' from Amersham Biosciences UK Limited/GE Healthcare Biosciences) was cultured according to the supplier's 30 instructions. Cells were transfected with plasmids (Figures 2a and 2b) encoding the PSLD-RFP fusion protein (SEQ ID NO: 1) using Fugene (Roche) according to the manufacturer's instructions. Cells were placed under 9 WO 2006/008547 PCT/GB2005/002890 hygromycin (125 pg/ml) and neomycin (500 pg/ml) selection, and surviving clones selected for further expansion. 5 Imaging of stable cell line expressing G1/S and G2/M sensors A stable U2OS cell line expressing Cyclin B1-EGFP and PSLD-J Red fluorescent fusion proteins was grown in 96 well plates in McCoys medium supplemented with 10% serum under standard tissue culture conditions. Cells were fixed in 2% paraformaldehyde, stained with Hoechst, and imaged using 10 an IN Cell Analyzer 1000 (GE Healthcare) with appropriate excitation and emission filters for blue (Hoechst), green (Cyclin B1-EGFP) and red (PSLD-J Red) fluorescence. Example 1 15 Images of the stable cell line cell line expressing Cyclin B1-EGFP and PSLD-J Red (Figure 3) show differential expression and localisation of the green and red fusion proteins between cells in different phases of the cell cycle. Determination of the presence or absence of green and red fluorescent 20 fusion proteins in the cytoplasm and nucleus of each cell allowed designation of cell cycle position according to the following scheme: 25 GI S G2 M Cytoplasm Green

-

+ + Red - + + + Nucleus Green

-

+ Red + - - + Experimental details relating to the production of stable cell lines expressing a polypeptide construct comprising a first detectable live-cell 30 reporter molecule linked to at least one cell cycle phase-dependent location 10 P %OPER\RAS\Claims3OI65999 ISPA doc-906/209 - 11 control element, the location of which construct within a mammalian cell is indicative of the cell cycle position, have been described in Applicant's copending US provisional patent application US60/645,968 entitled "Cell Cycle Phase Markers", the disclosure of which is incorporated herein by reference in its entirety. 5 The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. Although a few exemplary embodiments of this invention have been described, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially 10 departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the claims. Therefore, it is to be understood that the foregoing is illustrative of the present invention and is not to be construed as limited to the specific embodiments disclosed, and that modifications to the disclosed 15 embodiments, as well as other embodiments, are intended to be included within the scope of the appended claims. The invention is defined by the following claims, with equivalents of the claims to be included therein. The reference in this specification to any prior publication (or information 20 derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. 25 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other 30 integer or step or group of integers or steps.

Claims

1. A stable cell line expressing: i) a first polypeptide construct comprising a first detectable live-cell 5 reporter molecule linked to at least one cell cycle phase dependent location control element, the location of which said construct within a mammalian cell is indicative of the cell cycle position; and ii) a second polypeptide construct comprising a second detectable 10 live cell reporter molecule linked to a destruction control element wherein said second reporter is detectable in a mammalian cell at a predetermined position in the cell cycle, wherein said first and second reporter molecules are distinguishable from each other and the stable cell line can be used to determine the 15 cell cycle position.

2. The stable cell line of claim 1, wherein the cell cycle phase-dependent location control element is selected from the group of peptides consisting of Rag2, Chaf1B, Fen1, PPP1R2, helicase B, sgk, CDC6 or 20 motifs therein such as the phosphorylation-dependent subcellular localization domain of the C-terminal special control region of helicase B (PSLD).

3. The stable cell line of claim 1 or 2, wherein the destruction control 25 element comprises the Cyclin BI D-box.

4. The stable cell line of claim 1 to 3, wherein the first and second live-cell reporter molecules are selected from the group consisting of fluorescent protein and enzyme reporter. 30 12 WO 2006/008547 PCT/GB2005/002890

5. The stable cell line of claim 4, wherein said fluorescent protein is selected from Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Emerald and J-Red. 5

6. A stable cell line according to any preceding claim wherein the first reporter molecule is EGFP and the second reporter molecule is J-Red, or the first reporter molecule is J-Red and the second reporter molecule is EGFP. 10

7. A stable cell line according to any preceding claim wherein the first polypeptide construct comprises the phosphorylation-dependent subcellular localization domain of the C-terminal spatial control region of helicase B (PSLD) coupled to a red fluorescent protein (RFP), and the second polypeptide construct comprises 171 amino acids of the 15 amino terminus of cyclin B1 coupled to a green fluorescent protein (GFP) expressed under the control of the cyclin B1 promoter.

8. A method for determining the cell cycle position of a mammalian cell, said method comprising: 20 a) culturing a stable cell line according to claims 1-7; and b) determining the cell cycle position by monitoring signals emitted by the first and second reporter molecules.

9. A method for determining the effect of a test agent on the cell cycle 25 position of a mammalian cell, said method comprising: a) culturing a stable cell line according to claims 1-7; and b) determining the cell cycle position by monitoring signals emitted by the first and second reporter molecules wherein a difference between the emitted signals measured in the absence and in the 30 presence of said test agent is indicative of the effect of the test agent on the cell cycle position of the cell. 13 P:\OPER\RAS\ClaimS\30165989 ISPAdo-9/6/2f09 -14

10. A method for determining the effect of a test agent on the cell cycle position of a mammalian cell, said method comprising: a) culturing a stable cell line according to claims 1-7; b) determining the cell cycle position by monitoring signals emitted by 5 the first and second reporter molecules, c) comparing the emitted signals in the presence of the test agent with a known value for the emitted signals in the absence of the test agent; wherein a difference between the emitted signals measured in the presence of the test agent and said known value in the absence of the test agent is 10 indicative of the effect of the test agent on the cell cycle position of the cell.

11. A method for determining the effect of the mammalian cell cycle on a cellular process monitored by a process reporter which is known to vary in response to a test agent, said method comprising: 15 a) culturing a stable cell line according to claims 1-7 b) determining the cell cycle position by monitoring signals emitted by the first and second reporter molecules; and c) monitoring the signals emitted by the process reporter wherein the process reporter is distinguishable from the first and second reporter molecules, 20 wherein the relationship between cell cycle position determined by step b) and the signal emitted by the process reporter is indicative of whether or not the cellular process is cell cycle dependent.

12. A stable cell line according to any one of claims 1 to 7, or a method 25 according to any one of claims 8 to 11 substantially as herein before defined with reference to the Figures and/or Example.